Investigating Kissing to Duplex Dimer Transition Mechanism of HIV-1 SL1 by NMR.

Abstract

As a common feature to retroviruses, human immunodeficiency virus (HIV) packages two identical copies of its single stranded RNA genome (gRNA) that hold together at its 5’-end. gRNA dimerization is initiated by stem loop 1 (SL1), which consists of a highly conserved asymmetric internal loop and a GC-rich self-interacting palindromic apical loop that can drive dimerization by forming a meta-stable kissing dimer. During maturation, the kissing dimer undergoes a transition catalyzed by the viral nucleocapsid protein (NCp) into a thermodynamically more stable duplex. Both dimerization and structural isomerization between the kissing and duplex dimer are critical for viral replication and packaging. While SL1 and NCp have been the focus of many studies, the mechanism of the NCp dependent SL1 dimerization and isomerization remains poorly understood. This dissertation describes the characterization of SL1 structural dynamics, its Mg2+ and NCp binding properties, and the time-course of the kissing to duplex transition using high resolution nuclear magnetic resonance (NMR) spectroscopy. Initial studies were conducted on the conformational properties of the internal loop of SL1. Subsequently, we characterized the corresponding properties in kissing and duplex SL1 dimers along with their interaction with NCp and followed site-specifically the timecourse of the kissing to duplex transition using time-resolved NMR. We observe three types of motions that may promote conversion of the kissing dimer into its duplex form: (i) diffusion-limited nanosecond collective helix motions about the G-AGG internal loop that may help bring strands from distinct monomeric units into a proper register; (ii) a secondary structural switch occurring at μs-ms timescales which leads to partial melting of the upper-helix; and (iii) looping-in-and-out hinge motions of adenine residues in the apical loop that may help bring strands from different monomers into close spatial proximity. All three classes of motions are significantly dampened by Mg2+ which likely serves to make the transition strongly dependent on NCp. The NCp interacts with the internal loop and the apical loop of kissing dimer. Our results suggest that NCp stabilizes an alternative SL1 conformation, likely involving a quadruplex geometry, prior to transitioning in a single step into the duplex dimer.